The present invention relates a method and apparatus for the measurement of low-level laser-induced fluorescence in the field of cytofluorometry. Cytofluorometry has been greatly enhanced with the development of fluorescence dyes that are specific for deoxyribonucleic acid (DNA). These dyes permit the determination of the ratios of total DNA present in a cell population and, since the total amount of DNA doubles as a cell progresses through its proliferation cycle, the distribution of cell cycle positions that existed in a cell population is easily determined statistically. This is based upon the fact that, within necessary tolerances, the amount of dye bound to a cell is directly proportional to the amount of DNA present.
Fluorescence is the emission of light whose wavelength is different from that used to induce or excite the molecule of dye. Therefore, common fluorescence techniques require that the dye bind specifically to some component which is to be measured such as DNA. Such specificity is obtained by structurally modifying the dye molecule or coupling a dye molecule to another molecule that has the required specificity of binding. Another technique which utilizes fluorescence emission is based upon other properties of some dye molecules. When these molecules are in an aqueous environment, their fluorescense characteristics are distinctly different from that obtained in a hydrophobic environment. Since all biological membrances have hydrophobic regions, the amount and kind of fluorescence obtained following staining is related to the structural state of that membrane.
Several analytical techniques have been developed which exploit these various properties of fluorescent dyes. These techniques include photobleaching, fluorescence quenching, and shifts in fluorescence emission spectra. Problems are continuously encountered with these various techniques in that total fluorescence per cell, following any general technique with any particular dye, varies markedly and the variation is not quantitated for the expressed purpose of defining the degree or nature of cell cooperativity in a coordinating, interacting, cell mass.
Another fluorescence technique, stereological computer assisted cytofluorometry (SCAC) provides for the measurement of laser-induced cellular fluorescence in a cell or tissue mass (e.g. monolayer cell cultures or tissue sections). Dependent upon the nature of the fluorescent dye employed, the cellular response-density distribution profile will provide data of profound theoretical as well as practical significance. SCAC provides a technique for the quantitation of cell behavior and responses to drugs within the context of the cell mass or tissue. This has led to the development of new pharmacological parameters and is expected to lead to more refined and sophisticated parameters with which to study drug actions in the fields of cancer diagnosis, chemoprevention and chemotherapy, environmental and forensic toxicology, as well as basic biological sciences.
Currently there are two basic apparatuses for performing cytofluorometric studies. A fluorescence microscope such as the FACS .TM. series analyzer manufactured by Becton Dickinson provides observation of cells contained in a culture dish. The fluorescence microscope is highly accurate when analyzing a small area but cannot measure cell groupings larger than a culture dish without destroying the spatial relationship of cells. The second apparatus, a Flow Cytometer is designed to provide observation for a large number of cells, but the cells must be in suspension, thereby eliminating any possibility of obtaining spatial data. The CYTOFLUOROGRAF system from the Ortho Instruments Corporation is an example of a flow cytometer. The flow cytometer provides highly accurate information on the frequency distribution of fluorescence intensity in a randomly dispersed cell populations. However, no information is provided regarding the spatial relationship that may exist between cells in the tissue, tumor or culture prior to dispersal and staining.
Interpretation of frequency distributions of fluorescence intensity is seriously hampered by the fact that the response of the original cell population is rarely spatially homogenous. Heterogeneity of cell identity, morphology and drug responsiveness is commonly observed but not considered in current cytofluorometric analytic techniques. However, population heterogeneity is regarded by biologists as being an inherent quality of coordinating cell populations found in all animal tissues, tumors, primary cell cultures, and in rudimentary form, laboratory cell lines. The present invention, method and apparation, provides the ability to observe large areas of tissue, while maintaining full spatial relationships without the need for any special preparation.